高脂对大鼠肝星状细胞纤维化的影响

Effect of high fat on fibrosis in rat hepatic stellate cells

目的 研究高脂对大鼠肝星状细胞（HSC-T6）高迁移率族蛋白B1 （HMGB1）、α-平滑肌肌动蛋白（o-SMA）、基质金属蛋白酶2（MMP-2）表达的影响,探讨高脂对肝纤维化影响的机制.方法 体外培养HSC-T6,300μmol/L棕榈酸（PA）处理HSC-T6 24h,对照组采用等量牛血清白蛋白（BSA）处理HSC-T6 24 h,Western blot检测α-SMA、MMP-2的表达;剂量效应组分别用浓度为0 μ mol/L、100 μ mol/L、200 μ mol/L、300 μ mol/L、500μmol/L、1 000 μ mol/L的PA处理HSC-T6 24h;时间效应组用浓度为300μmol/L的PA分别处理HSC-T6 0h、4h、8h、12h、24 h、48 h,对照组采用等量BSA代替PA,Western blot检测HMGB1、α-SMA、MMP-2的表达;浓度为0 ng/ml、50 ng/ml、1oo ng/ml、200 ng/ml、500 ng/ml的重组人HMGB1 （rHMGB1）处理HSC-T6 48h,对照组采用等量磷酸盐缓冲液代替PA,Westem blot检测α-SMA、MMP-2的表达;PA组采用浓度为300 μ mol/L的PA处理HSC-T6 24 h,PA＋HMGB 1-siRNA组在下调HSC-T6细胞内HMGB1后加入300μmol/L的PA处理24h,空白对照组采用等量BSA代替PA,Westernblot检测HMGB1、α-SMA、MMP-2的表达.计量资料采用单因素方差分析,两两比较采用t检验.结果 （1）300μmol/L PA处理HSC-T6 24h,α-SMA、MMP-2的表达较0h明显增加（P＜0.05）;（2）不同浓度PA处理HSC-T6 24h,HMGB1、MMP-2的表达较未处理组（0μmol/L）明显增加（P＜0.01）,α-SMA的表达在PA浓度为200 μ mol/L、300μmol/L、500μmol/L、1 000μmol/L时出现明显增加妒＜ 0.01）;300 μ mol/L PA处理不同时间,HMGB1、α-SMA和MMP-2呈现不同程度的上升,并且在16 h、24 h、48 h较0h出现显著性增加（P＜0.01）;（3） 1oo ng/ml、200 ng/ml、500 ng/ml rHMGB1处理HSC-T6 48 h,α-SMA、MMP-2的表达较未处理组（0ng/ml）明显增加（P＜ 0.05）;（4）与PA组相比,PA＋HMGB1-siRNA处理HSC-T6 24 h,HMGB1、α-SMA和MMP-2的表达明显下降（P＜ 0.05）.结论 高脂可以通过上调HSC-T6中HMGB1的表达来促进α-SMA、MMP-2的表达,从而导致肝纤维化的发生.

Objective To investigate the effect of high fat on the expression of high-mobility group box 1 （HMGB1）,α-smooth muscle actin （α-SMA）,and matrix metalloproteinase-2 （MMP-2） in rat hepatic stellate cells （HSC-T6 cells）.Methods HSC-T6 cells were cultured in vitro and treated with palmitic acid （PA） at a concentration of 300 μmol/L for 24 hours,and the HSC-T6 cells in the control group were treated with bovine serum albumin （BSA） of the same volume for 24 hours;Westem blot was used to measure the expression of α-SMA and MMP-2.The HSC-T6 cells in the dose-effect group were treated with PA at concentrations of 0,100,200,300,500,and 1000 μmol/L for 24 hours;the HSC-T6 cells in the time-effect group were treated with PA for 0,4,8,12,24,and 48 hours;in the control group,PA was replaced by BSA of the same volume;Western blot was used to measure the expression of HMGB 1,α-SMA,and MMP-2.The HSC-T6 cells were treated with recombinant HMGB1 （rHMGB1） at concentrations of 0,50,100,200,and 500 ng/ml for 48 hours,and in the control group,PA was replaced by phosphate buffer of the same volume.Western blot was used to measure the expression of α-SMA and MMP-2.The HSC-T6 cells in the PA group were treated with PA at a concentration of 300 μmol/L for 24 hours;the HSC-T6 cells in the PA＋HMGB1-siRNA group were treated with PA at a concentration of 300 μmol/ L for 24 hours after HMGB1 in HSC-T6 cells was down-regulated;in the blank control group,PA was replaced by BSA of the same volume.Western blot was used to measure the expression ofHMGB1,α-SMA,and MMP-2.One-way analysis of variance was applied for continuous data,and the t-test was applied for comparison between two groups.Results （1） The expression of α-SMA and MMP-2 increased significantly after HSC-T6 cells were treated with PA at a concentration of 300 μmol/L for 24 hours （P 〈 0.05）.（2） Compared with the HSC-T6 cells in the untreated group （0 μmol/L）,the HSC-T6 cells treated with different concentrations of PA showed significant increases in the expression of HMGB1 and MMP-2 （P 〈 0.01）,as well as a significant increase in the expression of α-SMA at concentrations of 200,300,500,and 1000 μmol/L （P 〈 0.01）;the HSC-T6 cells treated with PA at a concentration of 300 μmol/L for different periods of time showed varying degrees of increase in the expression of HMGB1,α-SMA,and MMP-2,with significant increases at 16,24,and 48 hours （P 〈 0.01）.（3） Compared with the HSC-T6 cells in the untreated group （0 ng/ml）,the HSC-T6 cells treated with rHMGB 1 at concentrations of 100,200,and 500 ng/ml for 48 hours showed significant increases in the expression of α-SMA and MMP-2 （P 〈 0.05）.（4） Compared with the HSC-T6 cells in the PA group,the HSC-T6 cells treated with PA＋HMGB1-siRNA for 24 hours showed significant reductions in the expression of HMGB 1,α-SMA,and MMP-2 （P 〈 0.05）.Conclusions High fat can increase the expression of α-SMA and MMP-2 through up-regulating the expression of HMGB 1 in HSC-T6,and thus lead to the development of liver fibrosis.